U.S. patent application number 09/805549 was filed with the patent office on 2002-03-21 for compositions and methods for detecting redox-active molecules in solution.
Invention is credited to Creager, Stephen E..
Application Number | 20020034744 09/805549 |
Document ID | / |
Family ID | 26887840 |
Filed Date | 2002-03-21 |
United States Patent
Application |
20020034744 |
Kind Code |
A1 |
Creager, Stephen E. |
March 21, 2002 |
Compositions and methods for detecting redox-active molecules in
solution
Abstract
An electrochemical amplification scheme for detecting very small
amounts of redox-active molecules is disclosed. The reaction
involves "recycling" of oxidized analyte molecules by way of a
solution-phase electron exchange reaction with a sacrificial
electron donor. The scheme relies heavily upon the action of a
selective monolayer coating on the electrode that suppresses direct
oxidation of the sacrificial donor but facilitates the oxidation of
analyte molecules. The method is particularly useful for detection
of hydroxymethylferrocene at a dodecanethiolate-coated gold
electrode with ferrocyanide as the sacrificial electron donor.
Inventors: |
Creager, Stephen E.;
(Central, SC) |
Correspondence
Address: |
John E. Vick, Jr.
Dority & Manning, Attorneys at Law, P.A.
P.O. Box 1449
Greenville
SC
29602
US
|
Family ID: |
26887840 |
Appl. No.: |
09/805549 |
Filed: |
March 13, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60192211 |
Mar 27, 2000 |
|
|
|
Current U.S.
Class: |
435/6.11 ;
205/775; 205/777.5 |
Current CPC
Class: |
B82Y 30/00 20130101;
B82Y 5/00 20130101; G01N 27/3277 20130101; G01N 33/58 20130101;
G01N 2458/30 20130101 |
Class at
Publication: |
435/6 ; 205/775;
205/777.5 |
International
Class: |
C12Q 001/68; G01N
027/26 |
Claims
1. A method of detecting an analyte in solution, comprising: (a)
providing a solution having an analyte therein; (b) providing an
electrode in electrical communication with the solution; (c)
providing a sacrificial reagent in the solution; (d) transferring a
charge from the sacrificial reagant to the analyte; (e)
transferring a charge from the analyte to the electrode, thereby
generating an electrical signal; (f) repeating steps (d) and (e) to
amplify the electrical signal; and (g) measuring the amplified
electrical signal.
2. The method of claim 1 wherein the electrode further comprises a
coating layer adjacent to the electrode, the coating layer having
an upper surface in contact with the solution and a lower surface
in electrical communication with the electrode, the coating layer
being capable of conducting a charge from the analyte to the upper
surface to the electrode.
3. The method of claim 1 wherein the analyte is a derivative of
ferrocene.
4. The method of claim 3 wherein the analyte is
hydroxymethylferrocene (HMFc).
5. The method of claim 3 in which the sacrificial reagant is a
ferrocyanide.
6. The method of claim 1 wherein the signal is amplified by a
factor of at least one thousand.
7. The method of claim 1 in which the solution is provided in a
fluid delivery system that comprises a flowing stream.
8. The method of claim 7 in which the fluid delivery system further
comprises an analytical separation column and a detector.
9. The method of claim 2 in which the analyte is adsorbed upon the
upper surface of the coating layer at least during step (e).
10. The method of claim 2 in which the analyte is adsorbed upon the
upper surface of the coating layer during both step (d) and
(e).
11. The method of claim 2 in which the transfer of electrical
charges from the sacrificial reagent directly to the upper surface
of the coating layer is minimized.
12. A method of detecting relatively low levels of an analyte in
solution by amplifying amperometric signals, comprising: (a)
providing a solution having an analyte therein, the analyte
comprising a ferrocene derivative; (b) providing an electrode in
electrical communication with the solution, the electrode having a
coating layer affixed thereon, the coating layer having an upper
surface in contact with the solution and a lower surface affixed to
the electrode; (c) providing a sacrificial reagent in the solution;
(d) transferring a charge from the sacrificial reagent to the
analyte; (e) transferring a charge from the analyte to the upper
surface of the coating layer; (f) transferring a charge from the
coating layer to the electrode, thereby generating an electrical
signal; and (g) repeating steps (d)-(f) to amplify the electrical
signal.
13. The method of claim 12 further comprising the step of: (h)
measuring the amplified electrical signal.
14. The method of claim 12 further wherein the solution is employed
in a flowing stream.
15. The method of claim 12 in which the sacrificial reagent
comprises a ferrocyanide, further wherein the direct transfer of a
charge from the ferrocyanide to the upper surface of the coating
layer is minimized or eliminated.
16. The method of claim 12 in which the analyte is affixed to the
upper surface of the coating layer during steps (d) and (e).
17. The method of claim 12 in which coating layer is a
monolayer.
18. The method of claim 12 in which the ferrocene derivative
further comprises a tagged biomolecule, the biomolecule being
selected from the group of comprising: proteins, enzymes,
oligopeptides, oligonucleotides, and antibodies.
19. The method of claim 18 wherein the ferrocene derivative
comprises a ferrocene moiety attached to a strand of
deoxyribonucleic acid (DNA).
20. A bioaffinity ligand binding assay method that employs tagged
or labeled analytes of ferrocene to detect DNA, comprising: (a)
providing a labeled DNA strand having a ferrocene moiety; (a)
generating a capture DNA strand on a solid bead, the capture DNA
strand being combined with the labeled DNA strand; (c) exposing the
capture DNA strand to a target DNA strand; (d) displacing labeled
DNA strand from the capture DNA strand; (e) attaching the target
DNA strand to the capture DNA strand; and (f) detecting the
displaced labeled DNA strand by electrochemical detection
methods.
21. A method of determining the number of ferrocene tagged
biomolecules in solution at low detection levels, comprising (a)
providing at least one ferrocene group; (b) linking the ferrocene
group with a biomolecule; (c) tagging the ferrocene group; (d)
injecting the tagged ferrocene group, with attached biomolecule,
into a flow stream; (e) selectively detecting the tagged ferrocene
groups; and (f) determining the amount of biomolecules in solution.
Description
REFERENCE TO PREVIOUS APPLICATION
[0001] This application claims priority from previously filed
Provisional Application No. 60/192,211 filed on Mar. 27, 2000.
FIELD OF THE INVENTION
[0002] The invention is directed to a new method and system for
detecting redox-active molecules in solution at low concentration
levels.
BACKGROUND OF THE INVENTION
[0003] Amperometric detection of redox active molecules in solution
is used to detect very small amounts of a substance or chemical in
a solution via oxidation or reduction of that chemical, usually at
an electrode. This type of analysis is useful in forensic
chemistry, clinical chemistry, and many other applications in which
a trace amount of material is to be discerned in a solution.
However, such detection strategies are inherently limited by the
fact that each analyte molecule (i.e. the trace molecule being
detected) can accept or donate only a relatively small number of
electrons on oxidation or reduction. Because of this limitation,
and because of the noise inherent in measuring very small currents
(i.e. currents of less than a few hundred femtoamperes are
difficult to measure reliably), detection limits for amperometric
detection are often not nearly as low as would be desired.
[0004] Most strategies for improving the detection limits in such
processes involve some form of analyte recycling, either physically
(i.e. regeneration at a second electrode) or chemically
(regeneration via a coupled chemical reaction). Recycling
strategies in general are able to improve detection limits because
they amplify the signal, thereby increasing sensitivity without
increasing the background noise.
[0005] A prior art detection scheme that employs two electrodes
placed closely together has been used, in which the analyte is
regenerated by way of a second electrode that is placed in very
close proximity to the detection electrode. In this method,
amplification depends upon diffusion time between electrodes, and
the molecules must travel in a "circuit" at in order to carry out
the detection method. It is very difficult to achieve high
amplification in these systems, because the relatively short
residence time in the detector cell does not allow for many
recycling events. Amplification factors greater than 10.times. are
rare using such two electrode methods.
[0006] Another prior art detection scheme employs a chemical
reaction to regenerate the analyte. One characteristic feature of
most chemical recycling strategies is that they require the
presence of a reagant which can react with the oxidized or reduced
form of analyte chemically, but for which direct reaction at the
electrode is suppressed. The recycling scheme typically depends
upon the direct reaction of the reagant at the electrode being
inhibited, usually because it is kinetically too slow to occur as a
direct electrode reaction. It has often been necessary to use some
sort of catalyst (i.e. redox enzyme) to facilitate the recycling.
Unfortunately, reactions that depend upon catalysts are very
specific, and proceed too slowly, which can limit the amplification
factors that can be achieved using such methods.
[0007] What is needed in the industry is a relatively simple redox
amplification method which can provide a large signal enhancement
for amperometric electrochemical detection of redox molecules in
both quiescent solutions and flowing streams. A method that uses
relatively simple molecules and requires no enzyme catalyst would
be very desirable.
SUMMARY OF THE INVENTION
[0008] A new signal amplification scheme is employed in one aspect
of the invention for ultrasensitive amperometric electrochemical
detection of redox-active molecules in quiescent solution. The
invention may be employed in many different environments, including
in flowing streams. The method, in one embodiment, is based upon a
continuous regeneration of electrochemically oxidized analytes by
reaction with a sacrificial electron donor in solution. The method,
in one embodiment, utilizes a selective coating on the electrode.
The electrode may exhibit properties that facilitate relatively
facile electrooxidation of analyte. However, the invention also
serves to inhibit electro-oxidation of the sacrificial electron
donor on the electrode.
[0009] The method employs a solution having an analyte therein, and
an electrode in electrical communication with the solution. A
sacrificial reagant is also employed in the solution. A charge is
transferred from the sacrificial reagant to the analyte.
Furthermore, a charge is transferred from the analyte to the
electrode, thereby generating an electrical signal. The charge
transfer is repeated several times, facilitating amplification of
the signal, and the amplified electrical signal then may be
measured to determine the approximate concentration of the analyte
in solution.
[0010] In one aspect of the invention, a method is employed wherein
the electrode further comprises a coating layer adjacent to the
electrode, the coating layer having an upper surface in contact
with the solution and a lower surface in electrical communication
with the electrode, the coating layer being capable of conducting a
charge from the analyte to the upper surface to the electrode.
[0011] One embodiment of the invention employs an analyte which is
a derivative of ferrocene. One analyte that works well in the
method of the invention is hydroxymethylferrocene (HMFc). The
method may be employed with a sacrificial reagant comprising a
ferrocyanide. The method may amplify the signal by a factor of at
least one thousand, and in some embodiments, a detection of as
little as 60,000 or less injected molecules may be detected.
[0012] One embodiment of the invention employs a method of
detecting relatively low levels of an analyte in solution by
amplifying amperometric signals for a solution having an analyte
therein, the analyte comprising a ferrocene derivative. A
sacrificial reagant is employed in the solution, and a charge is
transferred from the sacrificial reagant to the analyte, and from
the analyte to the upper surface of the coating layer. Then, a
charge is transferred from the coating layer to the electrode,
thereby generating an electrical signal. By repeating this transfer
numerous times, an amplified signal is generated.
[0013] The method may be employed in which the ferrocene derivative
further comprises a tagged biomolecule, the biomolecule being
selected from the group of comprising: proteins, enzymes,
oligopeptides, oligonucleotides, and antibodies. In some
applications, the ferrocene derivative comprises a ferrocene moiety
which is attached to a strand of deoxyribonucleic acid (DNA).
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] A full and enabling disclosure of this invention, including
the best mode shown to one of ordinary skill in the art, is set
forth in this specification. The following Figures illustrate the
invention:
[0015] FIG. 1 is a schematic showing a prior art two electrode
method of detection in which a second electrode is placed in very
close proximity;
[0016] FIG. 2 shows a typical prior art method employing
sacrificial reagants and analyte;
[0017] FIG. 3 depicts a prior art system in which an analyte does
not participate directly in the charge-transfer reaction, but
rather serves as a catalyst to generate a quantity of a species
that can participate in a charge-transfer reaction;
[0018] FIG. 4 shows the electrochemical amplification method and
used with one embodiment of the invention in which a coating or
layer on top of an electrode receives an electrical charge from an
analyte;
[0019] FIG. 5 depicts a further system of the invention in which a
ferrocyanide is used as a sacrificial reagant, a ferrocene is
employed as an analyte, and the coating on the electrode is an
alkanethiol monolayer on gold;
[0020] FIG. 6 is a schematic showing one method in which the
invention may be employed wherein a separation column carries the
solution from a fluid delivery system to a detector cell;
[0021] FIG. 7 is a schematic showing an FIA apparatus that employs
an HPLC (high pressure liquid chromatography) system capable of
using a reference electrode and a working electrode, in one
embodiment of the invention;
[0022] FIG. 8 shows an example of a ferrocene tagged biomolecule
and system that can be used in the practice of the invention;
[0023] FIG. 9 shows one embodiment of the invention that employs a
DNA strand that is attached to a ferrocene cluster; and
[0024] FIG. 10 is a schematic of one embodiment of the invention
that uses a target DNA strand.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Reference now will be made to the embodiments of the
invention, one or more examples of which are set forth below. Each
example is provided by way of explanation of the invention, not as
a limitation of the invention. In fact, it will be apparent to
those skilled in the art that various modifications and variations
can be made in this invention without departing from the scope or
spirit of the invention. For instance, features illustrated or
described as part of one embodiment can be used on another
embodiment to yield a still further embodiment. Thus, it is
intended that the present invention cover such modifications and
variations as come within the scope of the appended claims and
their equivalents. Other objects, features and aspects of the
present invention are disclosed in or are obvious from the
following detailed description. It is to be understood by one of
ordinary skill in the art that the present discussion is a
description of exemplary embodiments only, and is not intended as
limiting the broader aspects of the present invention, which
broader aspects are embodied in the exemplary constructions.
[0026] A new signal amplification scheme for ultrasensitive
amperometric electrochemical detection of redox-active molecules in
quiescent solution and in flowing streams is described. The method
is based upon a continuous regeneration of electrochemically
oxidized analytes by reaction with a sacrificial electron donor in
solution. The method utilizes a selective coating on the electrode
that is chosen to have properties which allow for relatively facile
electrooxidation of analyte, but which also inhibits
electrooxidation of the sacrificial electron donor. Ultrasensitive
detection of hydroxymethylferrocene (HMFc) as a model analyte using
ferrocyanide as the sacrificial electron donor may occur at a
dodecanethiol-coated gold electrode, as one example.
[0027] Signal amplification factors of several hundred to several
thousand are obtained in flow-injection mode for analyte injections
in a concentration range between 10.sup.-4 and 10.sup.-7 M where
peaks can be discerned both with and without amplification. Even
higher amplification factors are estimated to be reached for
analyte concentrations below approximately 10.sup.-8 M, for which
peaks without amplification are typically undetectable.
Amperometric detection of 60 million injected HMFc analyte
molecules (corresponding to either a 10 L injection at 10.sup.-11 M
or a 1.0 mL injection at 10.sup.-13 M) is also possible using the
method in flow-injection mode, as further described below.
[0028] In this invention, a relatively simple redox amplification
scheme is provided that can provide large signal enhancements for
amperometric electrochemical detection of redox molecules in both
quiescent solutions and flowing streams. The method employs redox
molecules and requires no enzyme catalyst. As is usually the case
in such schemes, it does require that the medium include a quantity
of a sacrificial electron donor that will serve to recycle the
oxidized redox molecules via a solution-phase electron-exchange
reaction. A sample amplification scheme with hydroxymethylferrocene
as analyte and ferrocyanide as the sacrificial electron donor is
illustrated in FIG. 5. The electrode reactions in this scheme are
as follows:
HMFc.fwdarw.HMFc.sup.-
Fe(CN).sub.6.sup.4+HMFc.fwdarw.Fe(CN).sub.6.sup.3-+HMFc
[0029] The reaction between hydroxymethylferricenium and
ferrocyanide is approximately thermoneutral (the formal potentials
are less than 10 mV apart) but typically are relatively rapid since
both molecules are characterized by relatively rapid electron
self-exchange rate constants. Thus, this pair of reactants is well
suited to an electrochemical amplification scheme for detecting
ferrocene derivatives and ferrocene-tagged analytes.
[0030] In the application of the invention, it is possible to
enhance amperometric signals for redox active molecules without
enhancing noise levels. This enables very low limits of detection.
The method is also selective for specific categories of redox
active analytes. The invention may be employed in flowing streams,
or in other environments. Furthermore, the invention can be
employed to use tagged analytes, as further described below.
[0031] For comparison purposes, FIG. 1 shows a typical prior art
detection scheme that employs two electrodes placed closely
together. In this method, amplification depends upon diffusion time
between electrodes. It is very difficult to achieve high
amplification in these systems, because the relatively short
residence time in the detector cell does not allow sufficient time
for many recycling events to occur. Amplification factors greater
than 10.times. are rare using such two electrode methods.
[0032] In FIG. 2, a prior art method is shown that provides for
regeneration of the analyte by way of a chemical reaction with a
sacrificial reagant. The process usually requires that the
sacrificial reagant not itself be reactive with the electrode.
Often, such methods which have been used in the prior art require
enzyme catalysis, which can be slow and which often shows poor
reproducibility and poor long term stability.
[0033] FIG. 3 shows another prior art scheme that uses an analyte
that converts an oxidized reagant to a reduced reagant, so that the
reagant can work to carry out a reaction at an electrode surface.
Unfortunately, this sort of scheme requires a reaction that takes
place very close to the electrode surface, otherwise, the products
are transported away from the electrode. Most of such prior art
schemes also require enzyme catalysts which are undesirable for
reasons provided above.
[0034] In FIG. 4, a reaction methodology of this invention is shown
that employs a surface selective reaction 21. Surprisingly, it has
been discovered that by employing the analyte on or near the
surface of a coating layer 23 fixed to an electrode 24, it is
possible to facilitate the oxidation of the sacrificial reagant 25
on the analyte, but inhibit the oxidation of the reagant on the
coating layer 23. That is, the sacrificial reagant 25 will react
upon the analyte, but not upon the coating layer 23 of electrode
24, thereby providing the selectivity to amplify the signals
generated by transmission of the charge from the analyte 22 to the
electrode 24.
[0035] FIG. 5 shows one application of the invention that uses a
ferrocyanide as the highly charged sacrificial reagant and a
ferrocene as the analyte. A monolayer 28 is shown attached to the
electrode 29, and the reaction or charge transfer from the
ferrocyanide to the ferrocene, followed by charge transfer from the
ferrocene to the electrode 29. An "oily" monolayer 28 is comprised
of a series of hydrocarbon alkane chains 30a, attached to sulfur
groups 30b. The sulfur groups are directly adjacent to the
electrode 29.
[0036] The method illustrated in FIG. 5 requires that direct
ferrocyanide oxidation at the electrode be inhibited (i.e. the "X"
indicates that it is inhibited or minimized) but that direct
hydroxymethylferrocene oxidation at the electrode occurs relatively
rapidly. Coating the electrode with a self-assembled alkanethiolate
monolayer 28, as one option, may provide this surprising reactivity
pattern. Monolayers such as monolayer 28 can serve as excellent
barrier layers for preventing oxidation of highly charged and
well-solvated metal complexes such as ferrocyanide, but they are
usually not good barriers for preventing oxidation of neutral or
poorly-solvated molecules such as most ferrocene derivatives.
[0037] In a preferred embodiment, the selectivity of a
dodecanethiolate monolayer on gold is may be used to suppress the
direct oxidation of ferrocyanide, thereby enabling the signal
amplification scheme for hydroxymethylferrocene detection. Thus,
one preferred monolayer is dodecanethiolate, and one electrode that
is preferred is a gold electrode.
[0038] In FIG. 6, one application of the invention is shown
comprising a fluid delivery system 32. A sample comprising analyte
is injected at sample injection point 33, and flows along an
analytical separation column 34 to a detector 35. The detector 35
is operably connected to an output means, such as for example a
data output 36.
[0039] FIG. 7 shows a method of applying the compositions in the
practice of the invention in which an FIA apparatus ("FIA" denotes
flow injection analysis) is configured to supply a solution 42 by
way of a HPLC pump 41 (or other fluid delivery system) along line
41a. Injector 43 is the entry point for the analyte, and a guard
column 44 is used. A detector 45 is comprised of a reference
electrode 46 (which may be comprised of palladium, Pd) and a
working electrode 49 comprised of a metal such as gold. Further, a
flow exit 50 goes to waste. A potentiostat 47 is operably connected
to an output means such as recorder 48. In the practice of the
invention, a flow of about 1.0 ml/min with a 25 micrometer gap
width is possible. The working electrode 49 preferably has a
diameter of about 3 mm.
[0040] A new electrochemical amplification scheme for detecting
very small amounts of redox-active molecules is possible in the
application of the invention. The reaction involves "recycling" of
oxidized analyte molecules via a solution-phase electron exchange
reaction with a sacrificial electron donor. The method relies upon
the action of a selective monolayer coating on the electrode which
suppresses the direct oxidation of the sacrificial donor but
permits the facile oxidation of analyte molecules.
[0041] The scheme is demonstrated below for detection of
hydroxymethylferrocene at a dodecanethiolate-coated gold electrode
with ferrocyanide as the analyte, but other compounds could be used
in the application of the invention, and the invention is not
limited to the specific examples shown herein.
EXAMPLE 1
[0042] In one application of the invention, an analyte may be
measured by amplifying the electrical signal that is passed to the
electrode. A buffer (pH 5) used in both quiescent solution and
flowing stream experiments is composed of 0.10 M sodium perchlorate
(Alfa Aesar), 0.10 M acetic acid (Fisher) and 0.18 M sodium acetate
trihydrate (Alfa Aesar). Hydroxymethylferrocene (HMFc) was
purchased from Strem, a chemical supplier known to persons of skill
in the art. Sodium ferrocyanide decahydrate
(Na.sub.4Fe(CN).sub.6:10H.sub.2O) was obtained from Fluka Chemika
Company and mixed with the pH 5 buffer to make a 10.sup.-4 M
solution of ferrocyanide in the buffer. Due to the sensitivity of
ferrocyanide in solution to reaction with dissolved oxygen, this
buffer solution was prepared fresh daily. Mineral acids used to mix
the dilute aqua regia (1:3:4 HNO.sub.3(conc.):HCI(conc.):H.sub.2O
by volume) for etching electrodes were obtained from Fisher
Scientific. A 1-Dodecanethiol for monolayer preparation was
purchased from Aldrich Chemical. Reagents were used as received
from their respective manufacturers. All water for aqueous
solutions was deionized using a Barnstead Nanopure system to a
resistivity of about 17 megohm cm.
[0043] A 10 mM HMFc stock solution in ethanol was prepared by
adding a measured amount of solid HMFc to ethanol in a 10 mL
volumetric flask. 100 L of the 10 mM HMFc stock solution was
diluted with 900 L of buffer using two Wheaton Socorex Micropipetes
(100 L and 1000 L) to make a 10.sup.-3 M HMFc solution. The
solution was placed in a new 1.5 mL polypropylene microcentrifuge
tube (Fisher) and inverted and shaken 30 times to facilitate
mixing. The second dilution was performed by taking 100 L of the
10.sup.-3 M HMFc solution and adding 900 L of the buffer to make a
10.sup.-4 M HMFc solution. This process was repeated until all the
standards, 10.sup.-3 M to 10.sup.-13 M HMFc, were prepared. Two
sets of HMFc standards were made, one using pure buffer (no
ferrocyanide) and another using buffer containing 10.sup.-4 M
ferrocyanide. Ferrocyanide was included in both the running buffer
and the injected solution in FIA experiments to avoid small changes
in the ferrocyanide concentration as the injected plug passes
through the detector.
[0044] Electrodes for cyclic voltammetry were constructed of 0.127
mm Au wire (Alfa, Premion grade >99.999% pure), encased in epoxy
(Epon 825, Shell Chemical Company) that was crosslinked with
1,4-diaminocyclohexane (Aldrich Chemical Company) and hardened for
3 hours at about 80 degree C. The electrodes were then sanded and
successively polished by hand using 25, 5, and 1 micron alumina
with rinsing and a 1 minute etch in dilute aqua regia between each
polishing step. The 3 mm diameter gold electrode from the flowcell
was mounted in a Minimet 1000 Grinder/Polisher and polished using
the same process. This treatment produced an uncontaminated and
stable gold foundation upon which the monolayers could be formed.
After rinsing with water and isopropanol, the electrodes were
suspended in a 1.0 mM solution of alkanethiol in ethanol for 20-24
hours to form the monolayer.
[0045] Cyclic voltammetry was performed using a computer-based
CH-Instruments model 660 electrochemical workstation. A
three-electrode configuration with a platinum wire auxiliary
electrode and a Ag/AgCl/sat. A KCI reference electrode in pH 5
buffer electrolyte was used. The potential was swept over a range
from +0.0 V to +0.7 V at a scan rate of 0.1 Vsec.sup.-1.
[0046] The flow injection apparatus consisted of an ISCO model 2350
HPLC pump fitted with a Rheodyne model 7125 injection valve
connected to an ESA CouloChem II electrochemical detector with an
ESA model 5041 flow cell. The pump delivered buffer at a preset
flow rate of about 1 mL min.sup.-1. The buffer was continuously
degassed by bubbling with house nitrogen prior to pumping through
the FIA system.
[0047] Hydrodynamic voltammetry was performed in the flow cell by
setting the applied potential to the desired value, allowing the
current to stabilize and injecting a series of 10 L aliquots of a
1.times.10.sup.-6 M HMFc solution both with and without
1.times.10.sup.-4 M ferrocyanide present. The voltammetry was
conducted at both a bare gold electrode (etched for 1 minute in
dilute aqua) and at a dodecanethiol-coated gold electrode. The
etching step serves to clean the gold electrode prior to coating
with the dodecanethiol monolayer. It is a useful step, but not a
required component of this particular embodiment of the invention.
The potential was stepped in +0.1V increments for subsequent
injections. Current-time traces following injections were recorded
using a Lineseis Ly16100-11 chart recorder. Following each
injection at progressively positive potentials, a repeat injection
at +0.1 V vs. reference (an internal palladium-hydrogen electrode)
was made to establish whether the changes in response with
increasingly positive applied potential were reversible.
[0048] Current vs. time traces for different HMFc concentrations
were recorded for the series of injections of progressively more
dilute HMFc solutions in pure buffer (ferrocyanide free), and also
in buffer solutions containing 1.times.10.sup.-4 M ferrocyanide. A
short guard column (SiO.sub.2-packed) was included between the
injection port and the detector to help damp the pressure pulse
originating from the injection event. Detection was performed at an
applied potential of +0.4 V vs. reference for injections containing
between 10.sup.-4 M and 10.sup.-13 M HMFc for both buffer
series.
[0049] The ferrocyanide electrooxidation reaction was effectively
"blocked" by the monolayer. This effect reflects the fact that the
monolayer prevents close approach of ferrocyanide ions to the
electrode, which in turn causes the standard electron-transfer rate
constant for ferrocyanide to be greatly diminished relative to that
at an uncoated electrode. The slight rise in current at potentials
more positive than approximately +0.6 V probably reflects the onset
of long-range electron tunneling as a mechanism for oxidizing
ferrocyanide through the monolayer. This is an inherently slow
process, as indicated by the fact that such a large positive
overpotential is required to drive the oxidation. The oxidized HMFc
molecules have been recycled by the solution-phase
electron-transfer reaction between oxidized HMFc and
ferrocyanide.
EXAMPLE s2
[0050] In this example, ferrocene tagged biomolecules may be
detected at unusually low concentration levels. As seen in FIG. 8,
for example, biomolecules may be tagged by attachment to a
ferrocene derivative. Proteins, enzymes, oligopeptides,
oligonucleotides, antibodies and other compounds may be detected.
Tagging could be accomplished by linking a ferrocene group or other
redox molecule suitable for catalytic amplified detection to the
biomolecule via a tethering chain, for example the oligo-ethylene
glycol chain illustrated in FIG. 8. Alternatively, a cluster of
ferrocenes or other catalytic redox molecules could be linked via a
tethering chain to a biomolecule, as illustrated in FIG. 9 for a
cluster of ferrocenes linked to DNA. Tagging with ferrocene groups
would probably be accomplished prior to the biomolecule being
injected into a flow stream or separation column. With other
tagging molecules it is possible to accomplish tagging after the
molecules elute from a separation column. In this implementation of
the invention, selective detection of tagged molecules would be
accomplished by virtue of the fact that only the biomolecule(s) of
interest have been tagged.
EXAMPLE 3
[0051] In this example, amplified electrochemical detection of
ferrocenetagged bioaffinity agents could be used to detect the
binding of an (unlabeled) complementary molecule to the bioaffinity
agent via a change in the elution characteristics of the labeled
bioaffinity agent after binding of the complement. For example, if
the labeled bioaffinity agent is an antibody, then the elution time
on a chromatographic separation, or the electrophoretic mobility in
an electrophoretic separation, would be different depending upon
whether the labeled antibody was bound to it's complementary
antigen. Similarly, a labeled oligonucleotide would exhibit
different elution characteristics depending upon whether or not it
was bound to it's complement by sequence-specific base-pairing
hybridization. A change in elution characteristics on exposure to a
sample being tested would be indicative of the presence of
unlabeled complement in the sample.
EXAMPLE 4
[0052] In this example, amplified electrochemical detection of
ferrocenelabeled biomolecules is used to detect the binding of
target analyte molecules onto a surface via displacement of the
labeled molecules from a surface. The surface is pre-loaded with
weakly-bound labeled biomolecules, which are subsequently displaced
from their binding sited by the unlabeled target molecules when
they bind to the surface. The released labeled molecules are
ultimately detected in solution via amplified electrochemical
detection. The idea is illustrated in FIG. 10 for the specific case
of displacement of a ferrocene-labeled oligonucleotide that is
initially held via sequence-specific base-pair hybridization to a
complementary capture DNA strand that is immobilized onto a
surface, for example a solid bead. Upon exposure to a long strand
of target DNA that is complementary to the capture DNA strand, the
labeled DNA is released and subsequently detected via amplified
electrochemical detection, perhaps via injection into a flow stream
as described above.
EXAMPLE 5
[0053] In this example, amplified electrochemical detection is used
to detect simple redox-active molecules suitable for amplification
(i.e., a ferrocene that is not attached to a biomolecule) that are
released from an enclosed space (for example the inside of a
phospholipid vesicle). The objects that include the trapped redox
molecules (hereafter referred to as the vesicles) are captured at a
surface by a biospecific interaction, for example
base-pair-specific nucleic acid hybridization to nucleic acid tags
on the vesicle. Upon release of the redox molecules from the
vesicle, the redox molecules are detected with high sensitivity
using the electrochemical amplification method.
[0054] It is understood by one of ordinary skill in the art that
the present discussion is a description of exemplary embodiments
only, and is not intended as limiting the broader aspects of the
present invention, which broader aspects are embodied in the
exemplary constructions. The invention is shown by example in the
appended claims.
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